PROJECT SUMMARY: RNA modifications were recently rediscovered as essential regulators of gene expression. N6-methyladenosine (m6A) is identified as the most prevalent internal modification of eukaryotic RNA. The m6A modification controls RNA metabolism including RNA degradation and has been linked to human diseases such as obesity and cancer. However, its role in chronic pain including neuropathic pain is unknown. Our preliminary data suggest that a nerve injury-induced increase in dorsal root ganglion (DRG) FTO (fat mass and obesity-associated protein), a well-characterized RNA demethylase, may participate in neuropathic pain by reducing the level of m6A on Ehmt2 mRNA (encoding G9a, a histone methyltransferase), stabilizing nerve injury-induced G9a upregulation, and, consequently, silencing the expression of mu opioid receptor (MOR) in the injured DRG. Given that the transcriptional and translational changes in DRG gene expression following peripheral nerve injury participate in the development and maintenance of neuropathic pain and that G9a as a repressor of gene expression is a key endogenous contributor to neuropathic pain genesis, we propose that the increased FTO in the injured DRG contributes to neuropathic pain. In Aim 1, we will first determine whether pharmacological inhibition or genetic knockdown of DRG FTO attenuates nerve injury-induced pain hypersensitivity during the development and maintenance periods, and whether mimicking nerve injury-induced increase in DRG FTO leads to major symptoms of neuropathic pain in naive rats. In Aim 2, we will examine whether and how peripheral nerve injury upregulates the expression of FTO in the DRG. Time-dependent changes in the expression of Fto and its transcriptional activator Runx1 mRNAs and their proteins and in the level of m6A on specific RNAs in the DRG after peripheral nerve injury will be examined. We will also define whether nerve injury-induced up-regulation of DRG FTO is attributed to an increase in Runx1 in the injured DRG. In Aim 3, we will examine how DRG FTO participates in neuropathic pain. We will first observe whether FTO binds to Ehmt2 mRNA and whether this binding activity is increased in the injured DRG neurons after the fifth lumbar spinal nerve ligation (SNL). We will then define whether FTO contributes to the SNL-induced upregulation of Ehmt1 mRNA/G9a and the G9a-controlled downregulation of MOR by stabilizing the increased G9a expression in the injured DRG. Finally, we will determine whether blocking the SNL-induced increase in DRG FTO reduces the MOR-controlled primary afferent neurotransmitter release, restores the decrease of opioid analgesia, and attenuates opioid tolerance development. These studies will not only advance our understanding of posttranscriptional mechanisms of neuropathic pain, but will also open a door to develop a new strategy for the prevention and treatment of this disorder.